Information about most Saccharomyces cerevisiae genes, whether newly discovered or previously identified, is very limited. We have been using a transposon tagging strategy to analyze gene expression, protein localization, and gene disruption phenotypes for a large fraction of yeast genes with the ultimate goal of characterizing the yeast genome. Our transposons construct reporter gene fusions, epitope tag yeast proteins and large and small insertions in yeast genes. In the proposed funding period we plan to complete the collection of transposon insertions for every gene in the yeast genome. We will determine the subcellular localization of proteins by indirect immunofluorescence. Nuclear proteins will be analyzed to identify chromosomal proteins, and all of the binding targets of chromosomal proteins will be analyzed using a novel method involving chromatin immunoprecipitations and microarrays. We will generate a collection of yeast strains containing mutations in essential genes and attempt to characterize these strains using novel parallel screening methods and inducible protease cleavage methods. Finally, non-annotated reading frames and out- of frame coding sequences will be mutated and disruption phenotypes analyzed. We expect this project to have a significant impact on the scientific community. The information generated will be maintained in a database accessible by the Internet. Any researcher who identifies a yeast gene will be able to determine when the gene is expressed, the subcellular localization of the gene product and perhaps factors that regulate that gene's expression. Most importantly, plasmid alleles, libraries and yeast strains will be made available to all interested researchers. The technology proposed and the information gained will be applicable to the study of other organisms.